Liposomal transfection reagent

Human embryonic kidney cells (HEK293T), human myeloid leukemia mononuclear cells (THP-1), human hepatoma cells (Huh-7), mouse hepatocarcinoma cells (Hepa1-6), mouse mononuclear macrophages cells (Raw264.7) and mouse fibroblast cells (L929) were purchased from the China Center Type Culture Collection. HEK293T, Huh-7, Hepa1-6, Raw264.7, and L929 cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U/mL). THP-1 cells were cultured in RPMI1640 (Thermo Fisher Scientific, Waltham, USA) containing 10% FBS and 1% penicillin-streptomycin. All cells were grown in a 37°C cell culture incubator containing 5% CO₂.
For transfection of HEK293T, the cells were inoculated at 80% in the indicated culture dishes and used polyethylenimine (Polysciences, Philadelphia, USA) according to the manufacturer’s protocol. For macrophage cell lines, Raw264.7 and THP-1 cells were transfected with liposomal transfection reagent (Yeasen, Shanghai, China).

MLE-12 cells were seeded and incubated overnight before transfection. After mixing with Liposomal Transfection Reagent (Yeasen, China) in DMEM without FBS, penicillin or streptomycin for 25 min, the expression plasmid encoding CD146 or HIF-1α (Abmgood, China) was then transfected into MLE-12 cells at 90–95% density in DMEM for 48 h. The cells were harvested for further analysis. The blank vehicle plasmid was set as a control.

To assess the effect of APOL6 on cell viability, CCK8 experiment was performed with a CCK8 kit (Vazyme, Nanjing, China). The pancreatic cancer cell MIA PaCa-2 was obtained from ATCC and was seeded into 96-well plates at a density of 5 × 10³ cells/well. The next day, MIA PaCa-2 cells were transfected with APOL6 overexpression plasmid or empty vector plasmid using a liposomal transfection reagent (Yeasen, Shanghai, China). After culturing for 24 h, 48 h, and 72 h, the supernatant was replaced with fresh medium containing 10% CCK8 for additional 1 h. Relative cell viability was calculated as the absorbance of APOL6-transfected cells compared with vector-transfected cells.